Growth factors are polypeptides which stimulate a wide variety of biological responses (e.g., DNA synthesis, cell division, cell differentiation, expression of specific genes, etc.) in a defined population of target cells. A variety of growth factors have been identified including transforming growth factor-.beta.1 (TGF-.beta.1), TGF-.beta.2, TGF-.beta.3, epidermal growth factor (EGF), plotelet-derived growth factor (PDGF), fibroblast growth factor (FGF), insulin-like growth factor-I (IGF-I), and IGF-II.
IGF-I and IGF-II are related in amino acid sequence and structure, with each polypeptide having a molecular weight of approximately 7500 daltons. IGF-I mediates the major effects of growth hormone and thus is the primary mediator of skeletal growth after birth. IGF-I has also been implicated in the actions of various other growth factors, since treatment of cells with such growth factors leads to increased production of IGF-I. Both IGF-I and IGF-II have insulin-like activity (hence the name) and are mitogenic (stimulating cell division) for various types of cells involved in the growth and differentiation of skeletal tissues such as muscle and bone, as well as non-skeletal tissues.
IGF can be measured in blood serum to diagnose abnormal growth-related conditions, e.g., gigantism, acromegaly, dwarfism, various growth hormone deficiencies, etc. Although IGF is produced in many tissues, most circulating IGF is believed to be synthesized in the liver.
Unlike most growth factors, the IGFs are present in substantial quantity in the circulation, but only a very small fraction of this IGF is free in the circulation or in other body fluids. Most IGF is complexed with IGF-binding proteins. IGF in the blood is mainly complexed with IGFBP-3, the major circulating IGF-binding protein. Almost all IGF circulates in a non-covalently associated ternary complex composed of IGF-I or -II, an IGF specific binding protein termed IGFBP-3, and a larger protein termed the Acid Labile Subunit (ALS). This ternary complex is composed of equimolar amounts of each of the three components. The ALS has no direct IGF binding activity and appears to bind only a pre-formed IGF/IGFBP-3 complex. The ternary complex of IGF+IGFBP-3+ALS has a molecular weight of approximately 150,000 daltons. This ternary complex likely functions in the circulation "as a reservoir and a buffer for IGF-I and IGF-II preventing rapid changes of free IGF." Blum, W. F., et al., "Plasma IGFBP-3 Levels as Clinical Indicators", In Modern Concepts in Insulin-Like Growth Factors, E. M. Spencer, ed., Elsevier, N.Y., pages 381-393, 1991.
Having most circulating IGF in complexes is beneficial. Excess free IGF can cause serious hypoglycemia because IGF has insulin-like effects on circulating glucose levels. In contrast to the low levels of free IGFs and IGFBP-3, there is a substantial pool of free ALS in plasma which assures that IGF/IGFBP-3 complex entering the circulation immediately forms a ternary complex.
IGF Binding Proteins
IGFBP-3 is the most abundant IGF binding protein in the circulation. Recently, Wood et al. (Mol. Endocrin. (1988), 2:1176-85) and Spratt et al. (Growth Factors (1990), 3:63-72) described the cloning and expression of human IGFBP-3, whose structure is incorporated herein by reference. The gene for IGFBP-3 codes for 291 amino acids, the first 27 of which represent a characteristic signal sequence. Thus, the mature protein comprises 264 amino acids and has a predicted molecular weight of 28,749 (without glycosylation or other post-translational changes). When the human IGFBP-3 gene was expressed in Chinese hamster ovary ("CHO") cells and the conditioned culture medium was subjected to SDS electrophoresis and transferred to nitrocellulose membrane for ligand binding analysis, Spratt et al. reported "the presence of a 43-45 kd doublet, a 28 kd band and a minor 31 kd band" protein bands (p. 69), indicating there were post-translational changes. A side-by-side comparison revealed that the 43-45 kd doublet was also present in human serum.
FIGS. 12-15 show coding sequences and deduced amino acid sequences of IGFBP-3 suitable for various forms of use in the present invention.
It is unclear which tissue is the primary source of circulating IGFBP-3, although synthesis has been demonstrated in numerous cell types, including human fibroblasts, liver cells (most likely Kupfer cells) and osteoblasts. cDNA libraries that include the IGFBP-3 cDNA have been obtained from liver and other tissues. Vascular endothelial cells produce IGFBP-3 and may be the major source for systemic IGFBP-3.
IGFBP-3 has been purified from natural sources and produced by recombinant means. For instance, IGFBP-3 can be purified from natural sources using a process such as that shown in Martin and Baxter (J. Biol. Chem. (1986) 261:8754-60) IGFBP-3 also can be synthesized by recombinant organisms as discussed in Sommer, A. et al., In Modern Concepts of Insulin-Like Growth Factors, E. M. Spencer, ed., Elsevier, N.Y., pp. 715-728, 1991. This recombinant IGFBP-3 binds IGF-I with a 1:1 molar stoichiometry.
At least five other distinct IGF binding proteins have been identified in various tissues and body fluids. Although all these proteins bind IGFs, they each originate from separate genes and they have distinct amino acid sequences. Thus, the binding proteins are not merely analogs of a common precursor. For example, Spratt et al. compared the amino acid sequences of IGFBP-1, -2 and -3. Of the total 264 amino acids in the mature protein, only 28% of the amino acids are identical between IGFBP-3 and IGFBP-1, and 33% are identical between IGFBP-3 and IGFBP-2. Spratt et al. suggested that the similar portions of the binding proteins are the region(s) that bind IGF. Unlike IGFBP-3, the other IGFBPs in the circulation are not saturated with IGFs. All six known IGFBPs are reviewed and compared by Shimasaki and Ling, Prog. Growth Factor Res. (1991) 3:243-66.
Spencer et al. (1991) Bone 12:21-26; and Tobias et al. (1992) Endocrinology 131:2387-2392 report the stimulation of-bone formation by IGF-I.
The trabecular bone of rats, like that of humans, shows a coupling of bone formation to bone resorption, such that the increased resorption that occurs with estrogen deficiency entrains increased bone formation, which can be suppressed by inhibition of bone resorption. It has been established that in adult humans this coupling of formation and resorption involves a site specific sequence of events, in which bone resorption is normally followed, at the same site, by bone formation. Frost (1985) Clin. Orthop. Rel. Res. 200:198-225.
There is also some evidence that bone formation can occur without previous bone resorption, primarily in those situations where demands for mechanical support of the skeleton are increased (modeling). Parfitt, A. M., et al. (1984) Calcif. Tissue Int. 36:5123-5128.
The present invention offers in vivo single or combination therapy for stimulating new bone formation through the administration of the IGF/IGFBP complex or through the administration of the IGF/IGFBP complex and an agent which inhibits bone resorption. These combinations provide more effective therapy for prevention of bone loss and replacement of bone.